Bacterial signaling molecules that down-regulate pathogenic bacterial virulence properties

a signaling molecule and pathogenic bacteria technology, applied in the field of bacteria proteins, peptides and amino acids, can solve the problems of poor penetration at the tissue or biomaterial interface, inability to eradicate, and ineffective agents, and achieve the effect of increasing the proliferation ra

Inactive Publication Date: 2007-02-15
CHR HANSEN AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]FIG. 23 depicts suppressive effect of cell-free extracts (CFE) of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 on the in vitro proliferative responses of peripheral blood mononuclear cells (PBMC). PBMC obtained from 5 healthy controls were cultured with (+) or without (−) PMA, ionomycin and CFE. Results are expressed as mean optical density (OD) at 575 nm+SD, with higher OD corresponding to higher proliferation rate.

Problems solved by technology

However, these agents are often ineffective due to resistance of the offending organisms, inability to eradicate biofilms and poor penetration at the tissue or biomaterial interface.
>sp. These and other aerobic and anaerobic pathogens can cause severe morbidity and death amongst large patient populat
Implanted medical devices, such as heart valves and artificial veins and joints, are especially vulnerable to microbial biofilm formation and disease.
Urinary tract, vaginal and intestinal infections caused by E. coli can be serious, chronic or fatal.
Each year, an estimated 150 million cases of urinary tract infection (UTI) resulting in significant patient morbidity and billions of dollars in health care expenditures.
However, there is little known regarding the effects of Lactobacilli on the virulence of pathogens, such as urogenital pathogens.

Method used

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  • Bacterial signaling molecules that down-regulate pathogenic bacterial virulence properties
  • Bacterial signaling molecules that down-regulate pathogenic bacterial virulence properties
  • Bacterial signaling molecules that down-regulate pathogenic bacterial virulence properties

Examples

Experimental program
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Effect test

example 1

[0059] Virulence factors (VFs) produced by bacteria are exemplified below, in which the expression of virulence factors in a number of UPEC strains was confirmed (see Table 1). Oligonucleotide primers specific for the genes encoding VFs, such as type 1, P, F1 C and S fimbriae, haemolysin A, aerobactin, afimbrial adhesin I (afa I), cytotoxic necrotizing factors I and II (cnfs I and II), KII and KIII capsular proteins and 16S rRNA (control), were generated and PCR was carried out to screen for the presence of these genes. One PCR product for each VF was sequence verified. To confirm the PCR results, sequenced amplicons were also DIG-labelled and used as probes to screen genomic DNA of all the E. coli strains by dot-blot hybridization. The expression of haemolysin A and type 1 pili were also determined by plating on Columbia agar and haemagglutination assays, respectively.

TABLE 1Bacterial Strains Used in This StudyPreviouslyIdentifiedBacterial SpeciesStrainHuman OriginVirulence Facto...

example 2

[0061] To initially assess the effects of Lactobacillus secreted by-products on UPEC growth, differential antagonism and well diffusion qualitative assays were employed. The Lactobacillus strains Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 were grown as 1 cm wide lawns on BHIS agar for 48 hours. These lawns were then removed, perpendicular 1 cm lawns of each UPEC strain were plated across the original Lactobacillus streaks and plates were incubated 16-18 hours and the growth was assessed. Well diffusion assays involved testing spent culture supernatants (SCS) isolated from both Lactobacillus strains grown for 48 hours in BHIS broth on the growth of the UPEC strains. Lactobacillus SCS were pipetted into wells cut out of agar plates harboring surface lawns of the UPEC strains. The plates were incubated for 16-18 hours and the growth was assessed. Additionally, supernatants were either boiled for 10 minutes, neutralized to pH 7.0, treated with catalase or treated with proteinase ...

example 3

[0065] Using an experimental set up shown in FIG. 1, the ability of Lactobacillus-derived substances to affect the virulence of E. coli was demonstrated.

[0066] As shown in FIG. 1, the coculture of Lactobacillus and E. coli are separated by a 0.45 um membrane, which only allows molecules, not cells, to pass through. Thus, only by-products of the bacteria were able to induce the reaction or the bacterial “cross talk.” The first evidence of such bacterial “cross-talk” was obtained from a dot blot, which showed changes in S fimbrial expression caused by incubation with Lactobacillus GR-1. Similar changes were also observed from SQ-RT-PCR results, which showed that expression of VFs were altered, either up regulated or down regulated, when the E. coli strains were co-cultured with Lactobacillus GR-1 or RC-14. FIG. 4 to FIG. 9 illustrate the 2D protein gel results, which demonstrated that the expression of a uropathogenic E. coli VFs, such as fimbria, were altered when the E. coli were c...

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Abstract

This invention relates to composition and methods of employing said composition for treating or preventing microbial associated infections and diseases. More particularly the present invention relates to bacterial proteins, peptides and amino acids which are by-products of bacteria, in particular Lactobacillus and more specifically Lactobacillus strains GR-1 and RC-14, in compositions that can treat and prevent microbial-associated infections and diseases, by altering, for example, down-regulating, virulence properties of pathogenic organisms.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This is a continuation-in-part of International Application PCT / IB2004 / 003126, with an international filing date of Aug. 27, 2004, which claims the benefit of U.S. Provisional Application No. 60 / 498,960 filed Aug. 29, 2003.FIELD OF THE INVENTION [0002] This invention relates to bacterial proteins, peptides and amino acids which are by-products of Lactobacillus, preferably, Lactobacillus GR-1 and / or Lactobacillus reuteri RC-14, and which are not hydrogen peroxide, lactic acid, antimicrobial pheromones, mucin-inducer, biosurfactants or bacteriocins previously known in the art, in compositions that can treat and prevent infections and diseases, by altering (e.g., down-regulating) virulence properties of pathogenic organisms and / or enhancing host defenses. BACKGROUND OF THE INVENTION [0003] Microorganisms still represent one of the top three causes of death amongst humans and animals. The offending organisms can be bacteria, fungi, protozoa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/74A61K35/745A61K35/747A61K38/16A61L2/16A61P31/04C07K
CPCA61K35/745A61L2/16A61K35/747A61P31/04Y02A50/30
Inventor REID, GREGORCADIEUX, PETERMCCORMICK, JOHNDEVILLARD, ESTELLE
Owner CHR HANSEN AS
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